The present invention relates to methods for the purification of proteins using two-phase liquid-liquid extraction procedures. In particular, the methods of the invention are useful for the purification of human growth hormone, human growth hormone antagonists and their homologues.
Proteins are components of living organisms that play critical roles in phenomena like metabolism, gene expression, signal transduction, cellular and extracellular structures, and the like. Many proteins are useful for therapeutic or diagnostic applications; however, in order to utilize them therapeutically or diagnostically, it is necessary to prepare the protein of interest in pure form, i.e., without contaminants that could jeopardize the therapeutic or diagnostic goals and potentially put a patient""s health at risk.
The purification of proteins has long been a challenge, especially when large scale purification is sought as is typically required for therapy or diagnostics for large numbers of patients. However, even when small or medium scale purification of proteins is desired, a procedure that is fast and easy to carry out, while providing the protein of interest in sufficiently pure form and with high yields, is very desirable.
Human growth hormone (xe2x80x9chGHxe2x80x9d) and antagonists for hGH, i.e., growth hormone antagonists (xe2x80x9cGHAxe2x80x9d), are examples of proteins that are useful for a variety of therapeutic applications. hGH has been used for the treatment of hypopituitary dwarfism and all conditions resulting from low levels of hGH production, whether such condition is caused by genetic defect, injury or hypophysectomy. hGH has also been shown to improve the recovery of bum victims and other hospitalized patients. GHA, on the other hand, has been used to treat acromegaly, a form of gigantism caused by overproduction of hGH. Other possible medical indications of GHA are the prevention of retinopathy in diabetes patients, and the treatment of cancer patients with tumors overexpressing receptors that bind growth hormone (Clark et al., 1996, WO97/11178).
Even though the amino acid sequence of hGH is highly conserved, GH molecules from common domesticated animals do not work in humans. Only the protein extracted from the glands of higher primates functions in humans. As a consequence, hGH has originally been purified from pituitary glands recovered from cadavers. The general purification method involved extraction from tissues under mild alkaline conditions (Jones et al., 1979, J. Endocrinology 82:77-86) or with hot glacial acetic acid, followed by a series of fractionations by pH, ammonium sulfate, ethanol precipitation or polyethylene glycol precipitation (Nordisk Insulinlab, 1978, BE 857327) and a few chromatographic steps. It has also been purified from crude pituitary extract by immunoaffinity chromatography (Jonsdottir et al., 1986, Mol. Cell. Endocrinol. 46:131-135).
However, pituitary derived hGH has recently been linked to a certain number of cases of Creuzfelds-Jakob and has been removed from the market. To avoid this kind of problem, hGH had to be expressed in different systems. Early on, it was isolated from the supernatant of transformed monkey kidney cell cultures using hydrophobic interaction chromatography (Lefort et al., 1986, J. Chromatogr. 361:209-216). hGH does not undergo glycosylation and may therefore advantageously be expressed as a recombinant product in bacteria. In fact, hGH has been expressed in Bacillus subtilis (Franchi et al., 1991, J. Biotechnol. 18:41-54) and in Eschericia coli. Several extraction methods have been used, depending on the state of the expressed protein. hGH produced in the form of inclusion bodies was extracted by solubilization in elevated concentrations of chaotropic agents such as guanidine hydrochloride (Mukhija et al., 1995, Gene 165:303-306) or urea. Following refolding, the protein was then separated either by selective pH precipitation (Storrs et al., 1990, EP 445099) or directly loaded onto a chromatography column. hGH expressed in a soluble form has been purified from the periplasmic fraction of E. coli by anion exchange (Becker et al., 1986, FEBS Lett. 204:145-150) or from the crude cell lysate by immunoaffinity chromatography (Jonsdottir et al., 1986, supra) and ion exchange chromatography (Ettori et al., 1992, J. Biotechnol. 25:307-318).
Aqueous two-phase partitioning has been employed to separate proteins from cellular debris or to separate proteins from one another. Liquid-liquid extraction relies on the incompatibility between two polymers in aqueous solution, or one polymer and a salt present at high concentration. This incompatibility will result in the formation of two separate phases of very different compositions. The protein molecules will partition preferentially into one phase or the other, depending on their characteristics (Diamond et al., 1992, Advances in Biochem. Eng. Biotechn. 47:89-135).
Aqueous two-phase extraction procedures have been applied to the purification of hGH but such procedures always included chaotropic agents which denatured hGH (Builder et al., U.S. Pat. Nos. 5,407,810; 5,695,958; 5,723,310). However, the use of a chaotropic agent like, for example, urea, guanidine salts or thiocyanate salts to denature the hGH protein has critical disadvantages, especially when using the purification method on a large scale. For example, such chaotropic agents raise the cost of hGH purification, particularly as the chaotropic agent has to be available in a pure form. Also, once the hGH protein is denatured it must be refolded prior to most uses, especially prior to therapeutic uses, thereby increasing the necessary processing steps and potentially decreasing the amount of biologically active proteins.
The partition coefficient of hGH in a two-phase system was used to analyze the purity of hGH preparations following its purification and prior to its therapeutic use. However, no two-phase extraction procedures that do not use a chaotropic agent has been used to purify hGH or GHA (Heinsohn et al, U.S. Pat. Nos. 5,139,943; 5,151,358; Lorch et al., U.S. Pat. No. 5,328,841).
Thus, a simple, rapid and cost-effective method to isolate hGH and GHA using two-phase extraction without the need for a chaotropic agent is highly desirable.
The invention relates to aqueous two-phase extraction methods, wherein no chaotropic agent is used, for the purification of hGH and GHA, and homologues of either. The methods of the current invention are useful for the purification of recombinant hGH and GHA, and homologues of either, from any source.
In one aspect of the current invention, the methods provide for multi-phase extraction for the isolation of hGH, rGHA or a homologue of either. In a preferred embodiment of the current invention, the multi-phase extraction methods provide for two-phase extraction for the isolation of hGH, rGHA or a homologue of either. In another aspect of the current invention, the extraction methods provide for two-stage extraction for the isolation of hGH, RGHA or a homologue of either. Such extraction methods use a phase forming agent such as polyethylene glycol (xe2x80x9cPEGxe2x80x9d), a dextran, a ficoll, a Ucon or a poly(vinyl methyl ethyl ether), wherein each of the polymers can be used in various molecular weights for the isolation of hGH, rGHA or a homologue of either. In a preferred embodiment, PEG of molecular weights 2000, 4600 or 8000 is used as a phase forming agent. In another embodiment, a phase forming salt useful for the practice of the current invention is ammonium sulfate, sodium carbonate, potassium phosphate, magnesium sulfate or sodium sulfate for the isolation of hGH, rGHA or a homologue of either. In one aspect of the current invention, the extraction methods provided use Tris buffer, phosphate buffer, MES buffer, HEPES buffer or citrate buffer for the isolation of hGH, rGHA or a homologue of either. In one embodiment of the current invention, the extraction methods use as the source of the proteins to be purified any cell, tissue or animal, for example, animal cells, insect cells, plant cells, gram negative bacteria, gram positive bacteria, filamentous fungi, yeast or transgenic animals, or an extract or a supernatant derived from any one of the above. In a preferred embodiment, Escherichia coli cells are used. The extraction methods disclosed herein do not use a protein denaturing agent such as a chaotropic agent.
In a specific embodiment by way of examples, the extraction methods of the invention use PEG at concentrations of about 6% to about 15% weight per volume in the extraction mixture for the isolation of hGH, rGHA or a homologue of either. In addition, the extraction methods use ammonium sulfate at concentrations of about 6% to about 15% weight per volume in the extraction mixture for the isolation of hGH, rGHA or a homologue of either. The extraction methods are carried out at a pH of from about 6 to about 8 for the isolation of hGH, rGHA or a homologue of either.
The extraction methods disclosed herein can be carried out at small scale starting with less than about 10 liters of extraction mixture volume for the isolation of hGH, rGHA or a homologue of either. The extraction methods can also be carried out at medium scale of more than about 10 liters and less than about 100 liters of extraction mixture volume for the isolation of hGH, rGHA or a homologue of either. In a preferred embodiment, the extraction methods of the invention are carried out at large scale of more than about 100 liters of extraction mixture volume for the isolation of hGH, rGHA or a homologue of either.